Restriction Fragment Length Polymorphism Analysis of a Sodium

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Chapter 5

Restriction Fragment Length Polymorphism Analysis of a Sodium-Channel Gene Locus in Susceptible and Knockdown-Resistant House Flies, Musca domestica C. A . Bell, M. S. Williamson, I. Denholm, and A . L. Devonshire Institute of Arable Crops Research, Rothamsted Experimental Station, Harpenden, Hertfordshire AL5 2JQ, United Kingdom Resistance to D D T and pyrethroid insecticides i n the housefly (Musca domestica) often involves a common mechanism termed knockdown resistance (kdr). The voltage-sensitive sodium channel is generally regarded as the primary target for these insecticides, and has been implicated i n nerve insensitivity to these compounds conferred by kdr alleles. Part of the sodium channel gene, designated Msc, previously cloned from the housefly, was used to identify restriction fragment length polymorphisms (RFLPs) at this locus i n susceptible, kdr (resistant) and super-kdr (highly resistant) houseflies. These R F L P s showed tight linkage to resistance i n controlled crosses, providing the first genetic evidence that kdr, and hence pyrethroid mode of action, is closely associated with the sodium channel. We report here that sodium channel R F L P s at the Msc locus show much diversity amongst susceptible insects, but are strongly conserved in pyrethroid resistant laboratory and field populations. This further implicates the sodium channel as the site of resistance and suggests a common origin for the kdr and super-kdr alleles. Synthetic pyrethroid insecticides, developed from naturally-occurring pyrethrins, combine high insecticidal activity with low mammalian toxicity and lack of environmental persistence^. Their widespread use to control many agricultural and human health pests, combined with previous use of 1,1,1trichloro-2,-2-bis(4-chlorophenyl)ethane (DDT), which is thought to share the same mode of action and mechanism of resistance, has led to the rapid development of resistance i n many insect species (2,3). Several lines of evidence point to the nervous system as the primary target of D D T and pyrethroid action. In insects, the symptoms of pyrethroid poisoning are indicative of an action on the nervous system: rapid loss of coordinated 0097-6156/95/0591-0086$12.00/0 © 1995 American Chemical Society Clark; Molecular Action of Insecticides on Ion Channels ACS Symposium Series; American Chemical Society: Washington, DC, 1995.

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RFLP Analysis of a Sodium-Channel Gene Locus

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movement, periods of convulsive activity and ultimate paralysis. These features were recorded i n individual cockroaches using intracellular micro-electrodes (4), and implicated directly a modification of transient sodium ion conductance mediated by voltage-sensitive sodium channels as the primary cause of the observed effects on nerve action potentials. Later work on whole cell membranes showed that the kinetics of sodium channel activation, as well as inactivation, are different in the presence of D D T (5). Whilst other neuronal effects have been documented, effects on the sodium channel are considered to be the critical lesions mediating the action of these insecticides (6). D D T and Pyrethroid Resistance Resistance to both the rapid paralytic ("knockdown") and lethal actions of D D T and pyrethrins was first reported in the housefly (7), and shown to be controlled by a single gene (generally termed knockdown resistance or kdr) on autosome 3 (8). Subsequent work (9) confirmed these findings, and identified other putative allelic variants at this locus including the more potent super-kdr factor (10). Analogous mechanisms have been identified, though less well characterised, in several other pest species (reviewed in 11). The possible involvement of reduced neuronal sensitivity in kdr houseflies was suggested by the failure of synergists to increase the toxicity of D D T and pyrethroids i n kdr resistant houseflies, and by the location of the kdr gene on autosome 3, not known to carry genes conferring metabolic mechanisms of resistance to insecticides (9). Comparative studies of nerve preparations from susceptible and kdr type houseflies have shown that the latter survive longer periods of insecticide exposure or higher insecticide concentrations before nerve function is disrupted (12, 13). Two hypotheses have been advanced to explain the role of the sodium channel itself in the reduced sensitivity of the kdr phenotype. The first hypothesis proposes a reduced density of channels i n the nerve membrane, as has been observed in the nap* (no action potential, temperature sensitive) mutation i n Drosophila melanogaster. The presence of fewer channels was shown to be the cause of a low level of resistance to the knockdown effects of pyrethroids (14,15). This work prompted studies on the number of receptors binding to pyrethroids in kdr houseflies, but although one study reported a reduced sodium channel density associated with a kdr strain of housefly (16), others showed little or no difference in density (17-19). The alternative hypothesis is that kdr resistance reflects alterations i n the structure of the sodium channel protein, thereby reducing its affinity for D D T and pyrethroids. Until recently this hypothesis was only supported by indirect pharmacological evidence involving the effects of pyrethroids on the binding of known sodium channel ligands, demonstrating that D D T and neurotoxic pyrethroids enhance the binding of [ H] batrachotoxin to rat and mouse brain sodium channels (20, 21). 3

Clark; Molecular Action of Insecticides on Ion Channels ACS Symposium Series; American Chemical Society: Washington, DC, 1995.

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MOLECULAR ACTION OF INSECTICIDES ON ION CHANNELS

Cloning of a Sodium Channel Genefromthe Housefly A sodium channel gene was first isolated and sequenced from the electric eel (22), and subsequently from rat brain (23, 24), skeletal muscle (25, 26) and heart muscle (27). Two putative sodium channel genes have now been identified in Drosophila, termed DSC1 (28) and para (29, 30). By using homology to para, part of the equivalent housefly sodium channel gene encoding sequences towards the 3' (carbon-terminal) end of the gene was cloned and designated Msc (Musca sodium channel) (31). Restriction fragment length polymorphisms (RFLPs) at this locus were identified that differed between susceptible, kdr and super-kdr houseflies. Through a backcross strategy and discriminating dose bioassay, these sodium channel RFLPs were shown to segregate with resistance, providing clear genetic evidence that the sodium channel gene is tightly linked to knockdown resistance (31). Using a similar approach, Knipple et al (38) have recently confirmed this linkage for the kdr strain 538ge. Taken together, these results indicate that both the kdr and superkdr traits are located within one map unit of the sodium channel gene locus. The aim of the work described in the present paper was to study the sodium channel RFLPs in a wider range of susceptible and resistant strains to assess their potential for diagnosing and monitoring kdr resistance infieldpopulations. Materials and Methods Housefly Strains. The following strains were used and cultured using standard procedures. Cooper, a reference susceptible strain (9), lacking visible markers and homozygous for the wild type allele at the kdr locus; 579 and 530, homozygous for the kdr and super-kdr resistance alleles respectively and derived from the multimarked strains (53Sge and 3D respectively; see below) by repeated backcrossing to Cooper flies followed by re-selection for resistance (32); 538ge, homozygous for the kdr resistance allele and the recessive markers bwb (brown body) andge (green eye), all on autosome 3 (32); 3D, homozygous for the super-kdr level of resistance and possessing the recessive visible markers ac (ali-curve; autosome 1), ar (aristapedia; autosome 2) and ocra (ocra-eye; autosome 5) (32); A2, a wild-type strain collected in the Netherlands by F J . Oppennorth and homozygous for a third type of knockdown resistance (superfafrVu); 5640sel, homozygous for super-kdr levels of resistance (derived from strain 3D) and the visible markers ac, ar and ocra, and containing other uncharacterised metabolic resistance factors which interact with super-kdr to confer virtual immunity to pyrethroids; 171sel, a pyrethroid-resistant strain originatingfroma pig farm on the outskirts of Harpenden, UK, and maintained under heavy selection with permethrin in the laboratory, homozygous for knockdown resistance but also possessing other resistance mechanisms; Royston, collected in March 1993 from a large poultry rearing unit in North East Hertfordshire, UK, and exposed to heavy selection pressure in the field with pyrethroid, organophosphate and carbamate insecticides; Holtwood, also collected in March 1993 from a small pig rearing unit in South Bedfordshire, UK, only sporadically exposed to insecticides and for which no resistance problems had been reported.


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Bioassay Procedure. To assess resistance levels and attempt a diagnosis of any resistance alleles present in field populations, insects were bioassayed with D D T at two doses considered to be optimal for discriminating between susceptible and kdr homozygotes on the one hand, and kdr and super-kdr homozygotes on the other. These doses were 0.5 and 2.5 Mg / fly, both applied in conjunction with 1 Mg/fly PB (piperonyl butoxide) and 1 Mg/Ay F D M C (2,26&(4-chlorophenyl)-l,l,l-trifluoroethanol) to inhibit any other D D T resistance mechanisms present (33). Bioassays were performed on 3 to 4 day old milk-fed female adults, with up to 200 insects tested at each dose. Flies were anaesthetized with diethyl ether, treated topically with 0.5 /J1 of an acetone solution containing the appropriate dose of insecticide and synergists. Mortality was scored after holding for 24 h at 20°C. RFLP Analysis. Individual adultflieswere homogenised in 300 /il extraction buffer (0.1M Tris-HQ pH 8.8,0.1M EDTA, 1% SDS) using 15 ml microtubes with tight-fitting pestles (Kontes, supplied by Burkard Scientific UK) and incubated at 65°C for 30 min. 70 /ri 5M potassium acetate was added, incubated on ice for 30 min and centrifuged at 12000 rpm for 20 min. The supernatant was extracted with phenol and DNA precipitated with ethanol. D N A samples (5 Mg) were digested withiscoRI, separated on 0.8% agarose gels and transferred to nylon membrane using standard techniques (34). The membranes were hybridised to the ^-labelled cDNA insert of pSCP2 (31) in a solution containing 50% (v/v) formamide, 10 x Denhardts, 4 x SSPE, 0.5% SDS and 200 Mg/ml herring sperm DNA at 42°C for 16 h, and washedfirstin 2 x SSPE, 0.1% SDS at 60°C for 2 h then 0.5 x SSPE, 0.1% SDS at 60°C for 30 min. Membranes were exposed to X-ray film for 72 h. Results and Discussion RFLP Patterns of Laboratory Housefly Strains. RFLP patterns from standard 530 (super-kdr) 579 (fafr), and Cooper (susceptible) strains using the pSCP2' probe have been described previously (31). The bands identified in these strains were 8.8 and 3.0 kb for Cooper, 6.0 and 3.4 kb for 579 and >20, 15.5, 6.5 and 5.7 kb for 530. These three RFLP patterns are compared with representative patterns obtained from five further resistant laboratory strains in Figure 1. All of the additional strains contained, in various combinations, at least one of the bands present in 579 and 530 flies. No new bands were detected in these strains, nor were either of the bands characteristic of the susceptible Cooper population. Based on the limited numbers of insects examined (20-40), three strains (3D, 171sel and 5640sel) appeared homozygous for a single RFLP variant yielding a consistent pair of RFLP fragments. Two others (53Sge and A2) were, like 530, clearly polymorphic in this respect, possessing two allelic variants (each yielding a characteristic pair of RFLP fragments) present in homozygous or heterozygous condition. Strain 538ge was polymorphic with two variants yieldingfragmentpairs
20 with 6.5 kb and 15.5 with 3.4 kb. The first of these variants was also present in 530, and the second appeared identical to the more common variant in 53&ge. Strains 3D and 171sel were both homozygous for the 15.5 with 3.4 kb variant. 5640sel was homozygous for a variant yielding 15.5 with 6.5 kb bands, both of which were present but segregated independently in the 530 strain. In terms of individual bands, 15.5 kb was the most widely distributed and occurred i n six of the resistant strains examined, the only exception being strain 579. These results disclose a striking association between R F L P patterns, and the presence (though not necessarily the level) of knockdown resistance. Some similarities between laboratory strains were expected as a consequence of common descent from field populations in which kdr and super-kdr levels of resistance were first identified and characterised. Strain 579 was derived from 538ge by outcrossing to Cooper (to remove visible markers), followed by reselection with pyrethroids to recover the kdr resistance phenotype (32), whereas strains 5640sel and 530 were derived from 3D using other outcrossing procedures (A.W. Farnham, personal communication). However, strains A 2 and 171sel represented separate genetic lineages descended from field populations in Denmark and the U K respectively. The conservation of certain R F L P bands (those of 15.5 and 3.4kb in particular) i n these strains also cannot be attributed purely to coincidence; it implies a more fundamental genetic relationship between resistance alleles in these populations. Analysis of Field Populations. Once eggs had been collected from the Royston and Holtwood populations, insects from the original field collections were used for D N A analysis. R F L P patterns showed Holtwood to be highly variable at this locus; a representative sample is shown in Figure 2a. 14 different banding patterns were observed among the 16 Holtwood flies, 11 of which were putative heterozygote combinations. Many of the bands observed were shared by more than one individual, and, although it was difficult to ascertain whether any of these bands were the same as those previously observed, two individuals did seem to contain a band of 15.5 kb, similar to that found in 530 flies and other resistant laboratory strains. Royston flies showed much greater homogeneity of R F L P patterns, (Figure 2b). Among the 16 flies tested, 6 different patterns were detected, of which three patterns accounting for 12 individuals were clearly related to each other. One pattern was an apparent heterozygote combination with bands of >20, 15.5, 6.5 and 3.4 kb, and the other two being the corresponding homozygote patterns, with the >20 and 6.5kb bands and the 15.5 and 3.4 kb bands segregating i n combination. These banding patterns appeared identical to those observed i n the polymorphic A 2 strain (Figure 1). The other three banding patterns contained combinations of novel bands and ones already reported, but were not readily explained in terms of segregation of further allelic variants.



Figure 2. RFLP banding patterns of 12 representative individuals from each of the field collected housefly strains, A . Holtwood, B . Royston. Standards strains shown on the left are, Cooper, 579 (kdr) and 530

(super-kdr).


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Discriminating dose bioassays with D D T + F D M C + P B disclosed marked differences in resistance between the Holtwood and Royston populations, Table I. The lower dose of 0.5 /ig DDT/female (with both synergists applied a 1 Atg/fly) was one calculated, from previous experience, to kill susceptible and heterozygous genotypes but to allow resistant homozygotes (whether kdr or super-kdr phenotypes) to survive treatment. A s expected, all Cooper insects were killed by this dose, as were 100% of Holtwood flies. Mortality of 530 (super-kdr) and Royston insects was 0% and 0.4% respectively.

Table I. Mean percentage mortality of four housefly strains tested with D D T

—

1

Dose of D D T Strain

05 jig/fly

2S Mg/fly

Cooper

100

100

Holtwood

100

100

Royston

0.4

0

530

0

45 ± 9

| I

I

The higher concentration of D D T (2.5 /ig / fly) was one calculated to kill virtually all insects homozygous for kdr levels of resistance, but to allow at least 50% of super-kdr insects to survive exposure. The ca. 45% kill observed for 530 insects was i n keeping with this expectation. Failure to kill any Royston flies at this dose was indicative of virtually all insects being homozygous for knockdown resistance at least equivalent to, and perhaps exceeding the superkdr level of response. However, the response of Royston flies may have been modulated by the presence of the pen (reduced cuticular penetration) gene that alone confers very little resistance to D D T , but which may enhance the expression of any major resistance mechanism (including kdr) present (e.g. 35). Any metabolic resistance should have been suppressed by the added synergists. The pen factor was not present in the 530 strain. The Royston population was characteristic of others i n the U K subjected to intensive treatment with pyrethroids, causing strong selection for kdr and leading in most cases to severe control difficulties (36). The flies exhibited little variation i n R F L P banding pattern, and those bands present (especially 15.5 and 3.4 kb bands) resembled closely ones of resistant laboratory strains. The Holtwood population, in contrast, had rarely (if ever) been exposed to pyrethroids. N o resistance homozygotes were detected in bioassays and insects exhibited much variation i n R F L P banding patterns. Two of the sixteen flies tested showed a putative 15.5 kb band possibly indicative of heterozygotes possessing a single resistance allele. Since kdr resistance is effectively recessive


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in topical application tests, such insects would not have survived the discriminating dose bioassay. The conservation of R F L P banding patterns within and between resistant strains, compared with extensive polymorphism within the susceptible Holtwood population, mirrors results of work on two overproduced esterases (A2 and B2), generally associated, and implicated i n organophosphorus resistance in the mosquito, Culex pipiens (37). Resistance conferred by A2-B2 is now reported from four continents, and is a severe impediment to mosquito control i n some countries. Mapping of the usually very highly polymorphic B esterase region for six resistant strains from Asia, Africa and North America using 13 restriction enzymes produced virtually identical banding patterns. This signified that A 2 B2 overproduction had a single genetic origin, and has subsequently spread around the world through the passive transport of mosquitos on planes and boats (37). More extensive surveys of populations using further restriction enzymes and/or R F L P sites on the Msc gene could well implicate a similar phenomenon for knockdown resistance in houseflies. Results that are available support this hypothesis, and also imply that alleles conferring the different resistance patterns disclosed by bioassays (kdr, super-kdr and super-kdr^) arose as secondary modifications of the same original mutation. Sequencing work on susceptible and resistant strains is underway to explore these relationships in more detail. A clear-cut association between R F L P patterns and knockdown resistance implicates Msc still further as the gene responsible for this mechanism in houseflies. However, the question of whether such R F L P s constitute reliable genetic markers for resistance is still uncertain at present, since none of the bands identified occurred consistently i n all resistant populations. Studies on populations from a wider range of geographical localities, and on other restriction sites in the Msc gene (both now underway at Rothamsted) should clarify this issue further and may well identify markers even more closely conserved between resistant populations. Acknowledgments This work was supported by an A F R C studentship to C.A.B., and in part by the Ministry of Agriculture Fisheries and Food ( M A F F ) . The Resistance Group at Rothamsted is a member of the European Network for Insect Genetics in Medicine and Agriculture ( E N I G M A ) . Literature Cited 1. Elliott, M.; Janes, N.F.; Potter, C . Ann. Rev. Entomol. 1978, 23, 443-469. 2. Sawicki, R.M. In Progress in Pesticide Biochemistry and Toxicology. Hutson D . H . , Roberts T.R. Eds.; Wiley, New York, 1985, vol 5, pp 143-192. 3. Georghiou, G.P. In Managing Resistance to Agrochemicals; Green, M . B . ; LeBaron, H . M . ; Moberg, W.K., Eds; American Chemical Society, Washington, 1990, pp 18-41. 4. Narahashi, T. J. Cell. Comp. Physiol. 1962, 59, 61-65.


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Restriction Fragment Length Polymorphism Analysis of a Sodium

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